Antiviral Research 121 (2015) 120–131

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Antiviral Research

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Review

Chikungunya virus pathogenesis: From bedside to bench

http://dx.doi.org/10.1016/j.antiviral.2015.07.0020166-3542/� 2015 Elsevier B.V. All rights reserved.

⇑ Corresponding author at: Institut Pasteur, Biology of Infection Unit, Paris, France.E-mail addresses: therese.couderc@pasteur.fr (T. Couderc), marc.lecuit@pasteur.fr (M. Lecuit).

Thérèse Couderc a,b, Marc Lecuit a,b,c,d,⇑a Institut Pasteur, Biology of Infection Unit, Paris, Franceb Inserm U1117, Paris, Francec Paris Descartes University, Sorbonne Paris Cité, Division of Infectious Diseases and Tropical Medicine, Necker-Enfants Malades University Hospital, Institut Imagine, Paris, Franced Global Virus Network

a r t i c l e i n f o a b s t r a c t

Article history:Received 11 June 2015Accepted 4 July 2015Available online 6 July 2015

Keywords:Chikungunya virusArbovirusMosquito-borneEmerging virusArthralgia

Chikungunya virus (CHIKV) is an arbovirus transmitted to humans by mosquito bite. A decade ago, thevirus caused a major outbreak in the islands of the Indian Ocean, then reached India and SoutheastAsia. More recently, CHIKV has emerged in the Americas, first reaching the Caribbean and now extendingto Central, South and North America. It is therefore considered a major public health and economic threat.CHIKV causes febrile illness typically associated with debilitating joint pains. In rare cases, it may alsocause central nervous system disease, notably in neonates. Joint symptoms may persist for months toyears, and lead to arthritis. This review focuses on the spectrum of signs and symptoms associated withCHIKV infection in humans. It also illustrates how the analysis of clinical and biological data from humancohorts and the development of animal and cellular models of infection has helped to identify the tissueand cell tropisms of the virus and to decipher host responses in benign, severe or persistent disease. Thisarticle forms part of a symposium in Antiviral Research on ‘‘Chikungunya discovers the New World’’.

� 2015 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1212. Clinical presentation of chikungunya in humans: more than a benign disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

2.1. Chikungunya fever in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1212.2. Severe acute chikungunya in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1222.3. Chronic chikungunya in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

3. Experimental CHIKV infection in animal models mimicking some features of human chikungunya . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1234. Resolved and pending questions regarding the pathophysiology of CHIKV infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

4.1. Cell and tissues tropisms of CHIKV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

4.1.1. Acute phase of chikungunya disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1244.1.2. Severe acute chikungunya disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1254.1.3. Chronic phase of chikungunya disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

4.2. Host response to CHIKV infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

4.2.1. Innate response to CHIKV infection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1264.2.2. Adaptive immunity to CHIKV infection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1274.2.3. Immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

5. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

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1. Introduction

Chikungunya virus (CHIKV) is a member of the Alphavirusgenus, belonging to the Togaviridae family. Several alphavirusescause disease in humans. They are divided in two main phyloge-netically distinct groups: one that causes arthralgia and/or arthri-tis, mainly found in the Old World and which includes CHIKVand its closest relative Semliki Forest virus (SFV), O’NyongNyong, Ross River (RRV), Barmah Forest (BFV); and one that causesencephalitis, mostly found in the New World and that includeswestern equine encephalitis and Venezuelan equine encephalitisviruses (for review see Griffin, 2007). Sindbis virus (SINV), whichis geographically restricted to the Old World, is however phyloge-netically closer to the New World subgroup (Griffin, 2007). Uponmosquito bite, CHIKV induces an acute febrile illness typicallyaccompanied by severe arthralgia, which can last and relapse forweeks to months. CHIKV has been the cause of several outbreaksin Africa, from where it originates and was first identified in the50s, and in Asia. Since 2005, a new virus lineage (called IndianOcean Lineage, IOL) that originated from Africa, has caused a mas-sive outbreak in the Islands of the Indian Ocean, and reached India,South-East Asia, and also led to clusters of autochthonous cases inSouthern Europe. Since the end of 2013, a strain of CHIKV originat-ing from Asia has emerged the Caribbean and spread to South,Central and North America (Weaver and Forrester, 2015; Weaverand Lecuit, 2015).

Here we review the current knowledge on CHIKV infectionmainly obtained from the analysis of cohorts of human patientsand experimental animal models.

2. Clinical presentation of chikungunya in humans: more than abenign disease

CHIKV infects human through the bite of mosquito vectors andcauses disease called chikungunya, which means ‘‘walking bent’’ inMakonde, a language spoken in Austral Africa, where it was firstidentified (for review see Burt et al., 2012; Morrison, 2014;Staples et al., 2009; Suhrbier et al., 2012; Thiberville et al.,

Fig. 1. Successive steps of chikungunya virus infection in humans, based on human clinreplication at the inoculation site, in dermal fibroblasts. 3. Replication in target tissuesAdapted from Weaver and Lecuit (2015). Copyright Massachusetts Medical Society 2015

2013b; Weaver and Lecuit, 2015) (Fig. 1). Its symptoms are similarto classical dengue fever, except that they are associated withintense arthralgia, which is strongly predictive of chikungunya.The incubation period is short, lasting about 2–4 days (Fig. 2). Incontrast to dengue fever, asymptomatic infections are rare;roughly 3–25% of people with serological evidence of infectionhave no obvious symptoms. CHIKV infection is usuallyself-limited, non-fatal, with fever resolving within a few days.However, since the Indian Ocean outbreak in 2005–2006(Schuffenecker et al., 2006), the information available about theclinical characteristics of the human disease has significantlyincreased with the detailed clinical study of cohorts ofCHIKV-infected patients, notably in the Island of La Réunion, aFrench overseas department (Staikowsky et al., 2009). Previouslyunreported severe forms of CHIKV infection were observed, as wellas maternal–fetal transmission (Economopoulou et al., 2009;Gerardin et al., 2008; Rajapakse et al., 2010). The most notable clin-ical feature of chikungunya is related to the fact that, following theacute phase, joint symptoms may persist for weeks to months andeven years, with possibilities of relapses, leading to arthritis andsome cases destructive rheumatism, with pathogenesis has yet tobe fully understood (Queyriaux et al., 2008).

2.1. Chikungunya fever in humans

The incubation period ends with a sudden onset of high fever(>39 �C), back pain, myalgia, associated to severe and incapacitat-ing arthralgia, together with headaches, photophobia, and rash(for a review see above) (Figs. 1 and 2). The onset of fever coincideswith viremia, and blood viral load can rapidly reach up to 109 viralgenome copies per milliliter (Parola et al., 2006; Staikowsky et al.,2009) (Fig. 2). Viral replication triggers the activation of innateimmune responses, the hallmark of which is the production of typeI interferons (IFNs) (Schwartz and Albert, 2010).

A positive correlation between the intensity of viremia andacute illness has been observed. Actually higher viral loads havebeen found in hospitalized patients with comorbidity than thosewithout (Staikowsky et al., 2009) and is most often associated with

ical data and animal experiments. 1. CHIKV inoculation by mosquito bite. 2. Viral, with recruitment of inflammatory cells to infected tissues. 4. Joint inflammation.. Reproduced with permission.

Fig. 2. Timeline of infection, symptoms, and biomarkers. Shown is the chronology of viral replication in relation to clinical and biologic signs of disease, including thebiomarkers used in diagnostic assays to detect chikungunya virus infection. Adapted from Weaver and Lecuit (2015) and from Suhrbier et al. (2012). Copyright MassachusettsMedical Society 2015. Reproduced with permission.

122 T. Couderc, M. Lecuit / Antiviral Research 121 (2015) 120–131

clinical symptoms than lower viral load (Dutta et al., 2014).However, other studies have reported that the viral load of symp-tomatic individuals does not differ with clinical presentation orco-morbidity, although it tends to be higher than in viremicasymptomatic individuals (Appassakij et al., 2013; Thibervilleet al., 2013a). Several studies have also established that viral loadis higher in newborns and the elderly (Laurent et al., 2007;Staikowsky et al., 2009; Thiberville et al., 2013a).

Concomitant with viremia, the most common biological abnor-mality is leukopenia, and in particular lymphopenia (Staikowskyet al., 2009), which is more pronounced in patients with higher vir-emia (Borgherini et al., 2007). Other immunological markers asso-ciated with severe disease include notably high levels of type-IIFNs, IL-1b, IL-6, MCP-1 and TNFa (Kelvin et al., 2011; Ng et al.,2009; Venugopalan et al., 2014; Wauquier et al., 2011).Debilitating polyarthralgia is reported in the large majority ofsymptomatic patients, although children tend to display milderarthralgia (Jaffar-Bandjee et al., 2010). Joint pain is typically pol-yarticular, bilateral, symmetrical and affects mainly the extremi-ties (ankles, wrists, phalanges) but also larger joints (shoulders,elbows and knees) (Manimunda et al., 2010; Simon et al., 2007;Sissoko et al., 2010; Thiberville et al., 2013a). Joint symptoms canfluctuate in intensity, but do not usually vary anatomical location.Swelling may also occur in the interphalangeal joints, wrists, andankles, as well as pain along ligament insertions, notably in chil-dren. Arthralgia experienced by CHIKV-infected patients closelyresembles the symptoms induced by other viruses includingarthritogenic alphaviruses such as RRV and BFV (Jacups et al.,2008; Suhrbier et al., 2012). Myalgia is also frequently observed,its prevalence varying from one study to another (Mohd Zimet al., 2013; Staikowsky et al., 2009; Thiberville et al., 2013a), pre-dominantly in the arms, thighs and calves.

During the acute stage of CHIKV infection, rash occurs in 10% to40% of cases depending on the study (Borgherini et al., 2007;Economopoulou et al., 2009). It is characterized by transient mac-ular or maculopapular rash that involves mainly the extremities,but rarely the face, and lasts for 2–3 days. Children show a highprevalence of dermatological manifestations including hyperpig-mentation, generalized erythema, maculopapular rash and vesicu-lobullous lesions (Thiberville et al., 2013b; Valamparampil et al.,2009).

Rare ocular complications can occur during the acute illness, orwith a delay, including uveitis, iridocyclitis, and retinitis (Lalithaet al., 2007; Mahendradas et al., 2008).

Less frequently, symptoms include lymphadenopathy, pruritus,and digestive abnormalities, which are more common after viremia

has resolved (Staikowsky et al., 2009; Win et al., 2010; Wu et al.,2011).

Fever usually lasts less than a week, until viremia ends. This isthe time when patients mount anti-CHIKV adaptive immunity,characterized by the appearance of anti-CHIKV antibodies (Careyet al., 1969). Joint symptoms usually resolve within 1–2 weeks,but a large proportion of patients exhibit persistent or relapsingarthralgia that lasts for months or years (see below).

It is notable that disease severity may depend on hosts andvirus factors. The La Réunion isolate has been shown to replicateto higher level compared to both a West African lineage strain inrhesus macaques and an Asian lineage isolate responsible for therecent Caribbean outbreak in a mouse model (Messaoudi et al.,2013; Teo et al., 2015). This may correlate with differences in termsof acute disease severity as well as prolonged symptoms preva-lence associated with Indian Ocean vs. West Africa and Asia lin-eages, respectively.

2.2. Severe acute chikungunya in humans

Severe CHIKV disease in otherwise healthy individuals occursmainly in the extreme ages, in elderly patients and young children.Adults with severe disease usually display underlying condition,such as diabetes, alcoholic hepatopathy, stroke, epilepsy, hyperten-sion, or impaired renal function, which are independent risk factorsfor severe disease (Economopoulou et al., 2009). Severe disease canmanifest as encephalopathy and encephalitis, cardiovascular andrespiratory disorders, renal failure, hepatitis and myocarditis(Borgherini et al., 2007; Das et al., 2010; Economopoulou et al.,2009; Staikowsky et al., 2009).

Although CHIKV is not considered to be neurotropic, recent evi-dence suggests a neurological involvement in CHIKV infection,notably in infected neonates and young children and the elderly,who appear more prone to this complication. Most of the evidenceon CHIKV neurotropism stem from reports from the outbreaks dueto IOL CHIKV strains, over the past decade in La Réunion Island andIndia. Neurological presentations may include encephalitis,encephalopathy, acute flaccid paralysis, meningoencephalitis andGuillain–Barré syndrome (Economopoulou et al., 2009; Rampaland Meena, 2007; Singh et al., 2008; Wielanek et al., 2007). Theanalysis of a cohort of CHIKV-infected patients with CHIKV RNA-or anti-CHIKV-IgM positive cerebrospinal fluid (CSF) shows that42% of them fulfil the International Encephalitis Consortium crite-ria for encephalitis (Venkatesan et al., 2013), with an age distribu-tion curve exhibiting a U-shaped pattern with a very clear trendtowards the highest incidence at the youngest age (Gérardin

T. Couderc, M. Lecuit / Antiviral Research 121 (2015) 120–131 123

et al., under revision). In young children, including neonates, neu-rological complications include encephalitis, seizures and acuteencephalopathy.

Mother-to-child transmission of CHIKV infection was firstreported during the La Réunion outbreak, as a cause of severeneonatal disease, associated with neurological acute symptoms(Gerardin et al., 2008; Gerardin et al., 2014; Gupta et al., 2015;Ramful et al., 2007). The overall prevalence of maternal-fetal trans-mission is actually low (0.25% after 22 weeks) (Gerardin et al.,2008), and vertical transmission is observed exclusively innear-term deliveries in the context of intrapartum maternal vire-mia, with a rate that reached up to 50% during the La Reunion out-break (Gerardin et al., 2008). Whether host and viral genetic factorshave an impact on mother-to-child transmission remains to bedetermined. The preventive role of Caesarean section on transmis-sion is not precisely known. During the La Reunion outbreak, sev-ere illness was observed in 53% of infected neonates and mainlyconsisted of neurological signs (Gerardin et al., 2008).Importantly, CHIKV infection in the perinatal period can cause sev-ere disease with lifelong disability, as 51% of infected childrenexhibit global neurodevelopmental delay (Gerardin et al., 2014).

Detailed epidemiological investigations of maternal-fetal CHIKVtransmission, as well as human placental and experimental in vivoanimal studies have led to the conclusion that vertical contamina-tion most probably occurs as a consequence of passive transfer ofmaternal blood free CHIKV infectious particles through the placen-tal barrier via the physiological breaches that arise at the term ofpregnancy and during parturition, and which are known to leadto maternal-fetal blood exchanges (Couderc et al., 2008; Gerardinet al., 2008).

Hemorrhagic complications are exceptional, if they exist at all,and should therefore lead to the consideration of alternate diag-noses, such as a co-infection with DENV, or comorbidities such aschronic hepatopathy.

CHIKV-infected patients with severe disease often require hos-pitalization, in the context of their advanced age and loss of auton-omy. Deaths due to the infection were documented for the firsttime during the 2005–2006 outbreak in La Réunion(Economopoulou et al., 2009), and the most common causes ofdeath were heart failure, multiple organ failure, hepatitis, andencephalitis. In epidemics that have occurred since 2005, thecase-fatality rates were low, approximately 1 in 1000 (Borgheriniet al., 2007; Gerardin et al., 2008; Lemant et al., 2008; Tandaleet al., 2009). During CHIKV epidemics, a total of 260 excess deathswere reported in La Réunion island (mortality rate attributed tochikungunya 1/1000) and a total of 2944 excess deaths occurredin India (Ahmedabad) (mortality rate attributed to chikungunya0.8/1000) (Josseran et al., 2006; Mavalankar et al., 2007;Mavalankar et al., 2008; Pialoux et al., 2007). Most of the deathsoccurred in adults with underlying conditions, as well as, rarely,in neonates.

2.3. Chronic chikungunya in humans

In contrast to the other symptoms that manifest at the acutephase, joint pain may persist, and relapse, for weeks to monthsand even years (Ganu and Ganu, 2011; Schilte et al., 2013;Sissoko et al., 2009). Long-lasting symptoms are typically notobserved in dengue fever, although asthenia can be intense inthe days to weeks following the dengue fever. Chronic joint painfollowing chikungunya disease was first published in 1979(Fourie and Morrison, 1979) and has been massively reported dur-ing all recent epidemics that have occurred since 2005 in theIndian Ocean notably in the Island of La Réunion (Borgheriniet al., 2008; Hoarau et al., 2010; Javelle et al., 2015; Marimoutouet al., 2015; Schilte et al., 2013; Sissoko et al., 2009; Thiberville

et al., 2013a) or in metropolitan France for imported cases(Couturier et al., 2012; Larrieu et al., 2010; Simon et al., 2007), inIndia or Southeast Asia (Ganu and Ganu, 2011; Manimunda et al.,2010; Narsimulu and Prabhu, 2011; Win et al., 2010), in Italy(Moro et al., 2012) and more recently in the Caribbean (Foissacet al., 2015; Miner et al., 2015). Although the overall proportionof patients with chronic symptoms decreases over time afterCHIKV disease onset (from 100% to 88% during the first 6 weeks,to less than 50% after 3–5 years, with variable findings dependingon the studies), the time required for a complete healing of allsymptoms is still uncertain, as some infected individuals remainsymptomatic for years post-infection (Ganu and Ganu, 2011;Schilte et al., 2013; Sissoko et al., 2009).

Chikungunya chronic disease is characterized by persistent orrelapsing arthralgia usually located at the same joint sites thatwere affected during the acute phase, and may mimic rheumatoidarthritis (chronic inflammatory, and rarely erosive and evendeforming polyarthritis) (Ganu and Ganu, 2011; Javelle et al.,2015; Schilte et al., 2013).

In a recent 6-year retrospective study on a cohort of patients inLa Réunion, two main categories of post-chikungunya persistingrheumatic and musculoskeletal disorders were distinguished(Javelle et al., 2015): patients without previously defined arthritiswho correspond to 27% of patients, and who present with currentmusculoskeletal disorders (loco-regional or diffuse), and patientswith non-crystalline polyarthritis who represent 70% of patientsfulfilling the diagnostic criteria of rheumatoid arthritis, spondy-loarthritis or undifferentiated polyarthritis. The latter were referedto as patients with chronic inflammatory rheumatism (CIR).Among them, a minority (16%) had pre-existing CIR that immedi-ately exacerbated after CHIKV infection, while all the other devel-oped CIR following CHIKV infection. As CIR may progress in apotentially destructive disease, early disease management by aspecialized medical team is important. Notably, early treatmentwith anti-inflammatory and immunosuppressive drugs such asmethotrexate might prevent joint damage, although their safetyand efficacy remain to be validated in this context (Javelle et al.,2015).

It is noteworthy that joints that are already damaged by under-lying disorders, such as osteoarthritis seem to constitute preferen-tial sites for long-term pain (Sissoko et al., 2009). Of note, thelikelihood of developing persistent arthralgia is highly dependenton age (Javelle et al., 2015; Schilte et al., 2013).

This chronic disease has been reported to be associated withdetectable IL-17 and elevated serum levels of IL-6 and granulocytemacrophage colony-stimulating factor in patients at 2–3 monthsafter illness onset (Chow et al., 2011). However, in a study con-ducted in patients with chronic symptoms up to 36 months afterthe acute phase, no systemic biomarkers associated with chronicarthralgia nor biological markers typically found in autoimmuneor rheumatoid diseases were reported (Schilte et al., 2013).

Although the chronic disease generally causes less debilitatingpains than the acute disease, many patients still have a pro-nounced reduction in movement and quality of life and requirelong-term treatment (Couturier et al., 2012; Moro et al., 2012;Rosario et al., 2015; Schilte et al., 2013).

3. Experimental CHIKV infection in animal models mimickingsome features of human chikungunya

Animal models for CHIKV infection, including mice andnon-human primates have been used to study CHIKV-associatedpathologies (Gasque et al., 2015). A zebrafish animal model hasalso been developed and used to visualize CHIKV infection andinnate host responses to infection (Palha et al., 2013). However,

124 T. Couderc, M. Lecuit / Antiviral Research 121 (2015) 120–131

most studies have been conducted in mice, including investigationon CHIKV tissue and cell tropisms, as well as host responses toinfection.

Adult immunocompetent mice (wt mice) do not develop clinicalsigns following intraperitoneal, intradermal or intravenous virusinoculations (Teo et al., 2012). However, adult or 14-day-oldC57BL/6 mice develop viremia and pathological changes afterCHIKV inoculation via the subcutaneous route in the footpad,which are restricted to lesions in muscles of the infected foot, legswelling, edema with evidence of arthritis and tenosynovitis dur-ing the acute phase (Gardner et al., 2010; Morrison et al., 2011).Moreover, newborn and 14-day-old outbred mice, and 8–9 day-old C57BL/6 are susceptible to CHIKV infection, eitherthrough the subcutaneous route in the loose skin of the back, orin the thorax via the intradermal route, respectively (Coudercet al., 2008; Ziegler et al., 2008). They develop viremia and skeletalmuscle weakness that can be fatal in the case of younger mice andhistopathological analysis of the affected limb reveals myositiswith necrosis.

Adult mice with a complete deficiency in the type-I interferonreceptor (IFNAR�/�) develop a severe infection after intra-dermalCHIKV inoculation and to a lesser extent after ocular inoculation(Couderc et al., 2008; Couderc et al., 2012). The disease is charac-terized by muscle weakness of the limbs and lethargy, and is oftenfatal. Interestingly, adult mice with a partially abrogated type-Iinterferon receptor deficiency (IFNAR+/�) develop a mild diseasewithout viremia but with a low level of replication in musclesand joints, indicating that the gene copy number of the type-IIFN receptor influences viral load and tissue distribution, as wellas the severity of the disease (Couderc et al., 2008). Therefore,young age and inefficient type-I IFN signaling are major factorsof susceptibility to CHIKV severe disease in mice.

In contrast to adult wt mice, non-human primates are suscepti-ble to CHIKV infection and this susceptibility has been reportedsince 1967, when rhesus monkey were reported to be susceptibleto experimental infection with CHIKV as evidenced by a febrilereaction and high levels of circulating virus (Binn et al., 1967).More recently, studies performed in cynomolgus a (Macaca fascic-ularis) and rhesus (Macaca mulatta) macaques experimentallyinfected with CHIKV have shown that inoculation via the intra-venous route leads to systemic infection, with viremia levels ofup to 108 PFU/mL, even if the infectious dose inoculated is low(10 PFU) (Labadie et al., 2010; Messaoudi et al., 2013). With thehighest infectious dose (108 PFU), monkeys developed clinical neu-rological disease characterized by meningo-encephalitis (Labadieet al., 2010). At the peak of viremia, leukopenia including lym-phopenia is observed, similarly to lymphopenia observed inhumans at the acute phase of the disease, as well as markers oftype-I IFN response, inflammation, and cell immune activation(Labadie et al., 2010). In macaques, CHIKV targets joints, secondarylymphoid organs, liver, and, to a lesser extent, muscle and skin.Interestingly, long-term CHIKV infection can be observed, mainlyin secondary lymphoid organs in cynomolgus macaques (Labadieet al., 2010) and in aged rhesus macaques (Messaoudi et al.,2013). Thus, the CHIKV-infected monkey provides an animal modelto study these features of acute but also long-term evolution ofchikungunya.

The lower susceptibility of mice as compared to humans ormonkeys may involve species-specific factors. Actually, it has beenshown that the human autophagy receptor NDP52 interacts withthe CHIKV nonstructural protein nsP2, thereby promoting viralreplication in human cell cultures, whereas the NDP52 mouseortholog is unable to bind to nsP2 and to promote CHIKV infectionin mouse cell cultures (Judith et al., 2013). Thus, the absence of theproviral effect of NDP52 in the mouse may contribute to its lowerpermissiveness to CHIKV relative to humans. Whereas it is clear

that an increased neonatal susceptibility is also observed inhumans, the relevance of a type-I IFN defect and autophagy recep-tor NDP52 as a basis for severe infection in humans remains to bedemonstrated.

Studies performed in animal models experimentally infectedwith CHIKV have also contributed to decipher the pathophysiologyof the disease (see below).

4. Resolved and pending questions regarding the pathophysio-logy of CHIKV infection

Whereas the pathophysiology of infection with other alpha-viruses, such as SFV and SINV has been studied for several decades,the pathophysiology of CHIKV infection has been investigated onlyrecently. The studies performed in naturally infected human andexperimentally infected animal models have provided clues tothe cell and tissue tropisms of CHIKV, as well as host factors thatcontrol infection during the acute phase of infection.

4.1. Cell and tissues tropisms of CHIKV

4.1.1. Acute phase of chikungunya diseaseThe cell and tissue tropisms of CHIKV at the acute phase of the

disease have been investigated in humans and in animal models.Moreover, studies on CHIKV infection of cultures of primary cellsand cell lines have also contributed to the deciphering of the cellbiology of CHIKV infection.

In IFNAR�/� mice, CHIKV initially targets the liver, causes highviremia and replicates in connective tissues, particularly in theepimysium (also called muscle fascia) of skeletal muscle, inmyo-tendinous insertions of muscle and in joint capsules, and toa lesser extent in the perimysium and endomysium of skeletalmuscles (Couderc et al., 2008). Data in humans and infected muscleand joint tissues of mice showed that fibroblasts constitute majortarget cells of CHIKV at the acute phase of the infection (Fig. 1). Inmouse skeletal muscle, CHIKV can also be detected, albeit rarely, insatellite cells, consistent with a study performed on human mate-rial (Ozden et al., 2007). Thus, CHIKV infection pathophysiologylargely resembles to that of other arthritogenic alphaviruses, asthe connective tissues of joints and skeletal muscles, and tendonsare also the sites of replication of RRV and SINV (Heise et al.,2000; Morrison et al., 2006). Of note, both joint and muscle con-nective tissues contain a high amount of nociceptivenerve-endings, and their stimulation upon infection may accountfor the muscle and joint pain characterizing disease caused byalphaviruses associated with muscle and joint pathology(Couderc et al., 2009). The pain triggered by joint mobilizationmay also result from the infection of musculo-tendinous insertionssurrounding them.

Viral cell tropism in infected peripheral tissues of C57BL/6mouse neonates, including muscle and joints, is similar to that ofadult IFNAR�/� mice, with a pronounced tropism for fibroblasts(Couderc et al., 2008). A notable difference is the presence of severenecrotic myositis consistent with myofiber necrosis and inflamma-tion manifested by the presence of infiltrates of lymphocytes andmonocytes/macrophages. Similar data are found in young outbreadmice (Ziegler et al., 2008). Interestingly, in human adult musclebiopsies, myositis together with inflammatory infiltrates mainlyconsisting of monocytes/macrophages and T cells have beenreported (Ozden et al., 2007). Similarly to CHIKV in mouse neo-nates, RRV has been shown to induce myositis in mice (Heiseet al., 2000; Morrison et al., 2006; Ryman et al., 2000).

In the case of severe disease in mice, viremia is high and CHIKValso disseminates to other tissues, including skin, eye and the cen-tral nervous system (CNS) (Fig. 1). CHIKV targets fibroblasts of

T. Couderc, M. Lecuit / Antiviral Research 121 (2015) 120–131 125

deep dermis in the skin, as well as fibroblasts in the eye, includingthose of corneal and scleral stroma, corneal endothelium, ciliarybody smooth muscle stroma, iris and those between ocular musclefibers (Couderc et al., 2008; Couderc et al., 2012). In humans,CHIKV antigens have been found in fibroblasts of the same sites(Couderc et al., 2008; Couderc et al., 2012). Uveitis is due to inflam-mation of the uvea (iris, ciliary body, and choroid), and histologicaldata in CHIKV-infected ocular tissues may therefore provide a viro-logical basis for uveitis, the main ocular manifestation associatedwith CHIKV infection (Mahendradas et al., 2008; Rajapakse et al.,2010).

Together, these data demonstrate that infection of peripheraltissues responsible for symptoms in human in the context of acuteCHIKV infection, i.e. joints, muscle, skin and eye, is restrictedmainly to conjunctive tissues and the fibroblast as predominanttarget cell of CHIKV during acute infection. They also reveal thatCHIKV and other arthritogenic alphaviruses share some tissuetropism similarities.

In vivo findings are consistent with the in vitro observation thathuman and mouse primary muscle fibroblasts, as well as primaryhuman skin fibroblasts are susceptible to CHIKV infection (Coudercet al., 2008; Ekchariyawat et al., 2015). Fibroblasts derived from othertissues have also been shown to be permissive to CHIKV, includinghuman lung fibroblasts, primary human foreskin fibroblasts, andmouse embryonic fibroblasts (Schilte et al., 2010; Sourisseau et al.,2007). These cells, as well as primary human skin fibroblasts, producehigh level of IFNb triggered by MAVS activation upon CHIKV infection(Ekchariyawat et al., 2015; Schilte et al., 2010). It has been shown thatinfection of primary human skin fibroblasts triggers, in addition toIFNb, enhancement of IL-1b expression, maturation of caspase-1and expression of the inflammasome sensor AIM2 (Ekchariyawatet al., 2015). Moreover silencing of caspase-1 enhances viral replica-tion. This suggests that CHIKV-infected skin fibroblasts may con-tribute to a pro-inflammatory and antiviral microenvironment(Ekchariyawat et al., 2015).

The molecular basis for the prominent in vivo tropism forfibroblasts is unknown and may indicate that fibroblasts couldbe, relative to other cell types, either (i) in a hyper-permissive sta-tus regarding CHIKV entry/replication, and/or (ii) in ahypo-sensitive status to type-I IFN-mediated viral interference,making them a target of choice for CHIKV (Couderc et al., 2008).However, a study has shown that two human fibroblast cell lineswith different susceptibility to CHIKV infection failed to show thatdifferences in the primary type-I IFN and IFN-stimulated genes(ISG) responses to CHIKV were responsible (Thon-Hon et al.,2012). Alternatively, CHIKV cell-to-cell dissemination in fibroblastsmay be dependent on the particular structure of connective tissuesof dermis, joint capsules and muscles, that have in common theproperty to form a reticular network of cells interconnected bygap junctions (Langevin et al., 2004).

Beside fibroblasts, monocytes/macrophages are another celltype possibly targeted by CHIKV. Actually, CHIKV antigens weredetected in blood monocytes of acutely infected patients and mon-keys early after infection (Her et al., 2010; Roques and Gras, 2011).Primary human monocytes and macrophages could also beinfected with CHIKV, although with low efficiency (Sourisseauet al., 2007; Teng et al., 2012) and in vivo, viral antigens weredetected in monkey macrophages during the viremic phase ofinfection but also at later time points (6 weeks pi) (Labadie et al.,2010), suggesting that they may either be a site of CHIKV replica-tion, or of antigen clearance. These findings suggest that mono-cytes/macrophages might contribute to CHIKV physiopathology,although their contribution to viral load remains to be determined.

The susceptibility of monocytes/macrophages to CHIKV infec-tion has been further investigated using in vitro models. A studyrevealed that CHIKV infection of human macrophage cell lines

could be significantly increased by the presence of CHIKV-apoptotic blebs in the culture medium (Krejbich-Trotot et al., 2011).Another study showed that the murine macrophage cell lineRaw264.7 can be infected by CHIKV but that only a subset of cells issusceptible to infection and produce infectious particles, whereasthe others appears to be refractory to infection (Kumar et al., 2012).

Altogether these studies on monocytes/macrophages highlightthe lower susceptibility of these cells to CHIKV, as compared tofibroblasts, which may involve cell specific host cell factors deter-mining the susceptibility and/or resistance to infection. Comparedto monocytes, primary cultures of B and T cells were found not tobe susceptible to CHIKV infection in vitro (Her et al., 2010;Sourisseau et al., 2007; Teng et al., 2012).

Other in vitro studies have highlighted the specific susceptibil-ity or resistance of cells to CHIKV infection. CHIKV replicates inhuman muscle satellite cells but fails to infect differentiated myo-tubes (Ozden et al., 2007), according to in vivo findings.Interestingly, human primary keratinocytes can be infected byCHIKV but viral RNA synthesis is impaired such as de novo viralparticle are not produced, suggesting an intracellular block ofCHIKV replication in human keratinocytes (Bernard et al., 2015).According to this finding, the replication of CHIKV in keratinocytesin vivo has thus far never been reported. In vitro, mouse embryonicstem cells are susceptible to CHIKV infection and sense type I IFNs,but to a lower extent than CHIKV-infected fibroblasts. Moreover,they are deficient in type I-IFN expression, as compared toCHIKV-infected fibroblasts (Wang et al., 2014). Given the relativedeficiency of stem cells to produce and respond to type-I IFN, theymay constitute an important target for CHIKV in vivo, and mayhave a relevance for the long term consequences of infection.

4.1.2. Severe acute chikungunya diseaseIn the CNS of experimentally infected highly susceptible mice,

CHIKV targets the choroid plexuses, meningeal and ependymalenvelopes, but brain microvessels and parenchyma are spared inneonates, adult IFNAR�/� mice, as well as in adult infected mon-keys (Couderc et al., 2008; Ziegler et al., 2008). Primary mousechoroid plexus epithelial cells were highly susceptible to infection,while primary mouse brain microvessel endothelial cells were fullyresistant to CHIKV infection (Couderc et al., 2008). In humans,CHIKV and anti-CHIKV IgMs have been detected in the CSF of neo-nates and adult patients with CNS symptoms (Grivard et al., 2007).Altogether, these data suggest that CHIKV may infect and cross theblood–brain barrier at the choroid plexus and leptomeningeallevels and then infects CNS envelopes. In monkeys, high levels ofcytokines are found to be associated with encephalopathy(Labadie et al., 2010). Thus, the cytopathic effects induced ininfected cells of brain envelopes and the host responses triggered,may affect underlying neuronal cells, leading to the CNS signs andsymptoms associated with neurologic symptoms. In contrast toadult animals, a recent study has shown that CHIKV infectsparenchymal cells in neonatal/suckling mice but the nature ofthese cells is unknown (Dhanwani et al., 2011). A defective hostresponse may contribute to CHIKV neurotropism in neonates, aswell as to the generally higher susceptibility of neonates to severeCHIKV infection, in agreement with the fact that the neonatalimmune response is quantitatively and qualitatively distinct fromthat of adults (Adkins et al., 2004). In vitro studies show thatCHIKV replicates in mouse astrocytes and oligodendrocytes, andwith lower efficacy in neuronal cells but fails to infect microglia(Das et al., 2015). Further studies will be needed to fully decipherCHIKV tissue and cell neurotropism. In contrast, it is well estab-lished that New World alphaviruses cause encephalitis in humansand in animal models as a consequence of viral invasion of thebrain microvessels and parenchyma (Ferguson et al., 2015; Zacksand Paessler, 2010).

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Mother-to-child CHIKV transmission has been studied in exper-imental infection of pregnant animals. In pregnant viremic maca-ques and mice, transplacental transmission is not observed(Couderc et al., 2008; Chen et al., 2010). Investigation of mouseand human placentas from viremic mothers have shown that, incontrast to SFV and RRV, CHIKV does not directly infect trophoblas-tic cells and is therefore probably transmitted to neonates throughmaternal–fetal blood exchange during delivery, accounting for thelow frequency of mother-to-child transmission before near termdelivery (Aaskov et al., 1981a,b; Couderc et al., 2008; Milner andMarshall, 1984).

4.1.3. Chronic phase of chikungunya diseaseThe pathophysiology of CHIKV chronic disease remains poorly

understood and, to date, no animal model fully reproduces thechronic joint syndrome associated with many cases. In humans,patients with chronic CHIKV-induced arthralgia often have persis-tent virus-specific IgM (Borgherini et al., 2008; Malvy et al.,2009), that could result from continued exposure to CHIKV antigen.One study has provided evidence for persistence of viral antigenand RNA in synovial tissue from a patient with chronic arthralgiafor 18 months after CHIKV infection (Hoarau et al., 2010). In animalmodels, persistence of viral RNA has been shown both in mice andin monkeys. In mice, viral RNA can be detected in joint-associatedtissues for at least 16 weeks following footpad injection and is asso-ciated with histopathological evidence of joint inflammation(Hawman et al., 2013; Poo et al., 2014b). Interestingly, studies inCHIKV-infected macaque have demonstrated that viral RNA per-sists in lymphoid organs and liver and, to a lesser extent, in muscleand joints of macaques, and identified macrophages as the maincellular reservoirs during the late stages of CHIKV infection (Chenet al., 2010; Labadie et al., 2010), suggesting that macrophages playalso a role in chronic disease. Viral RNA persistence in both onehuman case and CHIKV animal models raises the question of therole of CHIKV RNA in joint inflammation and injury, as it has beenshown that double stranded RNA, a product of viral genome repli-cation, is arthrithogenic (Zare et al., 2004).

In addition to IgM, patients with chronic joint pains can displayelevated IL-6 levels (Chaaitanya et al., 2011; Chow et al., 2011;Reddy et al., 2014). For RRV, it has been shown that infection ofosteoblasts results in increased of IL-6 together with a change ofthe ratio of Receptor Activator of Nuclear Factor-KappaB Ligand(RANKL) to osteoprotegerin (OPG), which have been implicated inbone disease including arthritis and osteoporose (Chen et al.,2014). In mouse models, infectious RRV is detected in bones and areduction of bone volumes is observed in association with a disrup-tion of the ratio of RANKL/OPG. Importantly, the bone loss caused byRRV infection in mice, as well as the changes in the ratio RANKL/OPGare prevented by IL-6 inhibition (Chen et al., 2014). These findingssuggest a mechanism involving direct infection of osteoblasts toexplain joint pathologies during infection with RRV and possiblyCHIKV (Burt et al., 2014). As for RRV, pre-existing arthritis is exacer-bated by CHIKV infection (Javelle et al., 2015) and the inflammatoryresponse during alphavirus infection in the joint is similar to that inrheumatoid arthritis, with a similar pattern of leukocytes infiltra-tion, cytokine production and complement activation (Burt et al.,2012; Nakaya et al., 2012; Scott et al., 2010). Deciphering the viraland immune mechanisms leading to joint pathologies is a healthconcern, notably in the case of pre-existing arthritis given the rela-tively high incidence of bone disease in the general population.

4.2. Host response to CHIKV infection

Studies in humans and animal models have shown that innateimmunity, and particularly the type-I IFN response, is crucial for

restricting virus replication during the acute phase of infection.Moreover, as mentioned above, antibodies against CHIKV are pre-sent in the serum of infected humans at the end of viremia.

4.2.1. Innate response to CHIKV infectionAlphaviruses, including CHIKV, are long known to be strong

inducers of type-I IFN and sensitive to type-I IFN responses(Farber and Glasgow, 1972; Glasgow et al., 1971; Grieder andVogel, 1999). More recent studies in patients in La Réunion alsoreported that CHIKV infection elicited high levels of IFN-a in theserum and its concentration correlated with viral load (Schilteet al., 2010; Wauquier et al., 2011). Similar to human patients,the acute disease in monkeys is associated with substantiallyincreased type-I IFN concentration in the plasma (Labadie et al.,2010). As in mammals, CHIKV infection triggers a strong type-IIFN response, critical for survival in CHIKV-infected zebrafish(Palha et al., 2013).

Recently, the roles of type-I IFN in CHIKV infection and itsrelated antiviral pathways have been deciphered. As mentionedabove, adult IFNAR�/� mice are fully susceptible to sever CHIKVinfection, in contrast to adult wt mice. Interestingly, it has beenshown that this absence of susceptibility upon intradermal infec-tion of wt mice is due to the very early control of CHIKV infectionby type-I IFN at the site of injection in the skin. Actually, infectionis rapidly controlled by IFN-b secreted locally by infected dermalfibroblasts, which leads to antiviral response at the site of injection(Schilte et al., 2010). It is notable that no other cell type, includinghematopoietic cells, is infected at the injection site in wt adultmice.

The role of type-I IFN in CHIKV pathogenesis has been investi-gated further in humans and in mouse models, as well as in humancells. In mice, it has been demonstrated that CHIKV is controlled bythe direct action of type-I IFN on nonhematopoietic cells (Schilteet al., 2010), and nonhematopoietic cell-derived type I-IFN is suffi-cient to control CHIKV infection (Schilte et al., 2012). Moreover, theproduction of type I-IFN by nonhematopoietic cells acts via anMAVS-dependent signaling pathway likely triggered by activationof host sensors (RIG-I and/or MDA5) for CHIKV RNA in infectedfibroblasts (Rudd et al., 2012; Schilte et al., 2010; Schilte et al.,2012; White et al., 2011). It has also been shown that IRF-3 orIRF-7 expression in either hematopoietic or nonhemotopoietic cellcompartments is capable of inducing an antiviral response.Moreover, IRF-3 or IRF-7 signaling is sufficient to control CHIKVin adult mice, whereas both transcription factors are required inmouse neonates (Schilte et al., 2012). These findings indicate thatIRF-3 and IRF-7 play an essential role in the control of neonatalCHIKV infection in mice and highlight an age-dependent redun-dancy for IRF-3 or IRF-7 in adult but not newborn animals.

Type-I IFN is able to trigger the activation of a specific signaltransduction pathway leading to induction of ISGs that are respon-sible for the establishment of an antiviral state. ISGs that have beenfound to exert an antiviral role against CHIKV in vitro and/orin vivo include ISG15, ISG20, P56, ZAP, OAS3 and Viperin (Brehinet al., 2009; Lenschow et al., 2007; MacDonald et al., 2007;Werneke et al., 2011; Zhang et al., 2007).

The role of monocytes/macrophages in CHIKV disease has alsobeen investigated in the acute phase of the disease. Studies withnon-human primate and mouse models also suggest that viralreplication in joint tissues leads to the recruitment of inflamma-tory cells, with monocytes, macrophages, and natural killer cellsbeing the major inflammatory cell types (Gardner et al., 2010;Labadie et al., 2010). Consistent with the role of monocytes/-macrophages in inflammation during the acute disease are studiesshowing that macrophage depletion in mice reduces foot swellinginduced by injection of CHIKV in the footpad. Consistent with thisobservation, treatment with Bindarit, an inhibitor of monocyte

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chemoattractant protein-1 (MCP-1) chemokine (CCL2) production,blocks monocytes recruitment and reduces joint inflammation inmice (Chen et al., 2015; Gardner et al., 2010; Rulli et al., 2011).However, the recruitment of monocytes/macrophages also appearsto be critical for preventing excessive pathology and resolvinginflammation, as CHIKV-infection in CCR2�/�-deficient mice witha defective CCL2 receptor results in more severe disease due toan excessive recruitment of neutrophils rather than monocytes/-macrophages to the inflamed joint (Poo et al., 2014a).

These data provide evidence that monocytes/macrophages areinvolved in the pathophysiology of CHIKV acute disease. As men-tioned above, some of them seem to be targeted by CHIKV.Further studies will be required to clarify the relationship betweentheir role as target cells and as innate immune cells together withtheir role in inflammation during the acute phase of the disease.

Autophagy has been shown to be an innate mechanism involvedin host response to CHIKV infection. Actually, Atg16L(HM) mice,which display reduced levels of autophagy, showed a higher sensi-tivity to CHIKV-induced apoptosis and exhibited increased lethal-ity, suggesting that inducers of autophagy inhibits apoptosis andmay limit the pathogenesis of acute chikungunya (Joubert et al.,2012).

Besides innate response, CHIKV infection also leads to a protec-tive adaptive immunity.

4.2.2. Adaptive immunity to CHIKV infectionAnti-CHIKV immunoglobulin M (IgM) and immunoglobulin G

(IgG) are detected in the sera of infected patients during the acutephase of disease (Kam et al., 2012; Mohd Zim et al., 2013;Nitatpattana et al., 2014; Panning et al., 2008). These antibodiesexhibit a high in vitro neutralizing activity and in vivo studies havedemonstrated the importance of anti-CHIKV neutralizing antibodiesin the protection against CHIKV infection (see below,Immunotherapy). The protective role of antibody in CHIKV infectionis also illustrated by the report that infected mice deficient for TLR3signaling develop a more severe disease than wt mice and synthe-size anti-CHIKV antibodies that exhibit a lower in vitro neutraliza-tion potency than those generated in wt mice (Her et al., 2015).

The role of B and T cells has been investigated in Rag1�/� mice,which lack T and B cells. After infection in the footpad, Rag1�/�

mice display higher viral levels in a variety of tissues than wt mice,suggesting that adaptive immunity controls the tissue specificityand helps clear CHIKV infection (Hawman et al., 2013). The roleof B cells was also explored in B cell deficient (lMT) knockout miceinfected with CHIKV in the footpad. In these animals, viremia per-sisted for over a year, indicating a direct role for B cells and anti-body in mediating CHIKV clearance (Lum et al., 2013; Poo et al.,2014b). Moreover, these infected mice exhibited a more severe dis-ease than wt mice during the acute phase. The roles of T cells wereexplored in adult mice deficient for T cells and infected in the foot-pad. Interestingly, it was found that CHIKV-specific CD4+ but notCD8+ T cells are essential for the development of joint swellingwithout any effect on virus replication and dissemination, suggest-ing T cells are involved in inflammation (Hawman et al., 2013; Teoet al., 2013). This corroborates observations made from humanmuscle biopsies where T cells, but not B cells, were detected(Ozden et al., 2007).

The importance of B and T cells in protection against CHIKVinfections has also been demonstrated by vaccine studies(Mallilankaraman et al., 2011). Actually, vaccination againstCHIKV is able to induce a strong CD8+ T cell-mediated cellularresponse as well as a humoral response that protects mouse andnonhuman primate models against a lethal challenge(Mallilankaraman et al., 2011; Partidos et al., 2011).

Finally, the role of host age on the T and B cell responses hasbeen investigated in rhesus macaques. Interestingly, aged animals

have delayed and/or reduced T and B cell immunity compared toadult animals (Messaoudi et al., 2013). Moreover, while adult ani-mals are able to control viral infection, aged animals show persis-tent virus in the spleen. These data support clinical findings ofCHIKV susceptibility in elderly humans and provide evidence thatan effective T and B cell responses against the virus are required forpreventing persistent CHIKV infection.

4.2.3. ImmunotherapyThe protective effect of passive immunization against alpha-

viruses, including Venezuelan Equine Encephalitis virus, SINVand SFV, was long ago demonstrated in mouse models a long timeago (Boere et al., 1983; Mathews et al., 1985; Schmaljohn et al.,1983). In recent years, passive immunization has been investigatedin mouse models susceptible to CHIKV infection. The first studyreporting the use of passive immunization against CHIKV was per-formed with human polyvalent antibodies purified from humanplasma donors in the convalescent phase of CHIKV infection(Couderc et al., 2009). These antibodies exhibited strong neutraliz-ing activity in vitro and had a full protective efficacy in highly sus-ceptible mouse models: adult IFNAR�/�mice and neonatal C57BL/6mice. Moreover, they displayed a therapeutic efficacy in these ani-mal models when administrated after infection. Similarly, purifiedpolyclonal antibodies from monkeys immunized with a CHIKVvirus-like particle vaccine protected IFNAR�/� mice fromCHIKV-induced disease and death (Akahata et al., 2010).

As for neutralizing polyclonal antibodies, human and mouseneutralizing monoclonal antibodies have been shown to protectmice against CHIKV. Human neutralizing monoclonal antibodiesdirected against E2 or E1 significantly delay lethality ofCHIKV-infected mice, both in prophylactic or therapeutic settings(Fong et al., 2014; Fric et al., 2013). Similarly, a human neutralizingmonoclonal antibody directed against E2 protein is able to prophy-lactically protect adult C57BL/6 mice from viremia and foot swel-ling, and to therapeutically protect neonatal C57BL/6 mice fromdeath (Selvarajah et al., 2013). In other studies, mouse neutralizingantibodies directed against the E1 or E2 protein have been shownto provide both prophylactic protection from viremia and footswelling of adult C57BL/6 mice and from CHIKV-induced lethalityin adult IFNAR�/� mice (Goh et al., 2013; Pal et al., 2013).Interestingly, combinations of two neutralizing monoclonal anti-bodies administrated after CHIKV infection completely preventmortality of IFNAR�/� mice.

Altogether, these studies suggest that passive immunizationmay constitute an effective medical intervention for humans witha known exposure to CHIKV who are at risk for severe disease. Thisprophylaxis approach could thus be recommended especially dur-ing birth for neonates born to viremic mothers, who are at high riskof developing severe infection (Gerardin et al., 2008). The protec-tive role of anti-CHIKV hyperimmune human intravenousimmunoglobulins in neonates exposed to a high risk of severe formof CHIKV infection is currently under clinical investigation (seeClinicalTrials.gov number NCT02230163).

These data on the protective role of neutralizing antibodiesagainst CHIKV disease should be taken into account in the designof an efficient vaccine against CHIKV.

5. Conclusions

Repeated, massive CHIKV outbreaks of the last decade have elu-cidated many new facets of CHIKV infection, including detailedclinical analysis of chronic debilitating arthragia and severe dis-ease. Chikungunya can no longer be considered as a purely benign,self-limited disease. Studies performed in patients and animalmodels have provided the first data on the pathophysiology of

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infection and have shown the similarities and differences withother alphavirus, as well as with dengue virus infection.However, many questions remain to be resolved, particularly thesusceptibility and the role of immune cells in the acute and chronicdisease and the neurotropism of CHIKV infection. Increased basicand translational research, with access to tissues and cells frominfected patients, will be key to answer these questions and iden-tify all the host and viral factors involved in the susceptibility ofthe host, tissue and cell to CHIKV. This research will allow us tobetter prevent and treat the chronic and/or severe diseases causedby this emerging alphavirus.

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